Protozoan parasites are a serious health threat in many areas of the world. Trypanosoma cruzi (T. cruzi) is one such parasite that infects millions of individuals. Ten to thirty percent of individuals infected with T. cruzi develop chronic symptomatic Chagas' disease, which may in turn lead to heart disease and a variety of immune system disorders. T. cruzi infection has long been a public health problem in Central and South America. It is estimated that 18 million people worldwide are chronically infected with T. cruzi, but available drug treatments lack efficacy and often cause serious side effects.
The most significant route of transmission in areas where the disease is endemic is through contact with an infected triatomid insect. However in other areas blood transfusions are the dominant means of transmission. Accordingly, in order to inhibit the transmission of T. cruzi, it is necessary to develop accurate methods for both diagnosing T. cruzi infection in individuals and for screening blood supplies. Blood bank screening is particularly important in South America, where 0.1%-62% of blood samples may be infected and where the parasite is frequently transmitted by blood transfusion. Due to high flow of immigrants to the US from many Central and South American countries where T. cruzi infection is endemic, the US blood supply is becoming at high risk for contamination from T. cruzi infected blood donors. While there are a few tests available for diagnosing infection in individuals, there is currently no FDA approved test available in the US for blood donor screening for T. cruzi infection.
The diagnosis of T. cruzi infection has been problematic, since accurate methods for detecting the parasite that are suitable for routine use have been unavailable. During the acute phase of infection, which may last for decades, the infection may remain quiescent and the host may be asymptomatic. As a result, serological tests for T. cruzi infection are the most reliable and the most commonly used form of diagnosis. Such diagnoses are complicated, however, by the complex life cycle of the parasite and the diverse immune responses of the host. The parasite passes through an epimastigote stage in the insect vector and two main stages in the mammalian host. One host stage is present in blood (the trypomastigote stage), while a second stage is intracellular (the amastigote stage). The multiple stages result in a diversity of antigens being presented by the parasite during infection. In addition, immune responses to protozoan infection are complex, involving both humoral and cell-mediated responses to the array of parasite antigens.
While detection of antibodies against parasite antigens is the most common and reliable method of diagnosing clinical and subclinical infections, current tests for T. cruzi infection are generally insensitive, lack specificity, and are not suitable for screening of blood supplies. Most serological tests use whole or lysed T. cruzi and require positive results on two of three tests, including complement fixation, indirect immunofluorescence, passive agglutination or ELISA, to accurately detect T. cruzi infection. The cost and difficulty of such tests has prevented the screening of blood or sera in many endemic areas.
U.S. Pat. Nos. 5,876,734 and 6,228,601 disclose compositions useful for diagnosing Chagas' disease that comprise a non-repetitive region of the T. cruzi protein TCR27, and fusion polypeptides including such regions. U.S. Pat. No. 6,419,933 discloses a fusion polypeptide referred to as TcF that contains the four antigenic T. cruzi peptides PEP-2, TcD, TcE and TcLo1.2, together with methods for the use of the fusion polypeptide in the detection of T. cruzi infection. While TcF is highly reactive with T. cruzi-infected sera from South America, it exhibits low activity, and is occasionally negative with, Central American sera. U.S. Pat. No. 6,458,922 discloses an assay for T. cruzi infection that employs compositions comprising at least six antigenic T. cruzi peptides selected from the group consisting of: SAPA, CRA, FRA, TcD, Tc24, Ag39 and MAP. Published US Patent Application No. US-2004/0132077-A1 discloses recombinant polypeptides and fusion polypeptides (referred to as FP3, FP4, FP5, FP6, FP7, FP8, FP9 and FP10) useful for diagnosing T. cruzi infection. The disclosed fusion polypeptides comprise modified versions of previously identified T. cruzi epitopes, including TC27, TCR39, SAPA and MAP.